CN104255710B - A kind of method optimizing roxburgh anoectochilus terminal bud protocorms cryopreservation by vitrification effect - Google Patents

A kind of method optimizing roxburgh anoectochilus terminal bud protocorms cryopreservation by vitrification effect Download PDF

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CN104255710B
CN104255710B CN201410468248.0A CN201410468248A CN104255710B CN 104255710 B CN104255710 B CN 104255710B CN 201410468248 A CN201410468248 A CN 201410468248A CN 104255710 B CN104255710 B CN 104255710B
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protocorms
terminal bud
roxburgh anoectochilus
anoectochilus terminal
vitrification
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CN104255710A (en
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张荻
张亚非
任丽
陈冠群
王路尧
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Shanghai Jiaotong University
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Abstract

The invention discloses a kind of method optimizing roxburgh anoectochilus terminal bud protocorms vitrification ultra-low temperature preservation effect, for adopting the vitrification solution process roxburgh anoectochilus terminal bud protocorms containing carbon nanomaterial to improve its preservation effect, specifically comprise: preculture, the process of loading liquid, vitrification solution process and Liquid nitrogen storage step, wherein said vitrification solution contains 0.1 ~ 0.5g/L graphene quantum dot.Method disclosed in the present invention is remarkable to the preservation effect optimization of roxburgh anoectochilus terminal bud protocorms, plays facilitation by adding graphene quantum dot as allogenic material to the preservation of plant vitrification ultra-low temperature.

Description

A kind of method optimizing roxburgh anoectochilus terminal bud protocorms cryopreservation by vitrification effect
Technical field
The present invention relates to the preservation field of plant or its local, be specifically related to a kind of method optimizing roxburgh anoectochilus terminal bud protocorms vitrification ultra-low temperature preservation effect.
Background technology
Excised Embryos is the modern germ plasm resource Plantlet in vitro technology grown up the seventies in last century.Usually preserve in liquid nitrogen, be saved metabolism in Materials Cell and vegetative activity almost stops completely, be in metastable biological condition, reach the object of long-term conserving species matter, Excised Embryos is the medium-term and long-term preserving type uniquely not needing continuous subculture at present.Cryopreservation by vitrification cell or tissue is placed in the vitrification solution be made up of a certain proportion of permeability and impermeability protectant; make material and vitrification solution thereof under enough fast rate of temperature fall, be solidified into amorphous glassy state, and preserve at low temperatures with this glassy state.Vitrification is because of simple and quick, and cost is low, is suitable for preserving kind extensively, and preserve material genetic stability, the advantages such as preservation effect is good are nearly ten years for the prefered method of the medium-term and long-term preservation of fine germplasm resources.
Roxburgh anoectochilus terminal bud is the orchid family Anoectochilus Blume herbaceos perennial, among the people, there is extensive medical value, have the laudatory title such as " king of medicine ", " gold grass ", " god's grass ", " bird ginseng ", therefore set up the Excised Embryos technology of effectively preserving roxburgh anoectochilus terminal bud germ plasm resource most important.The orchid family is the first section in angiosperm, as a kind of important ornamental plants resources, in the evolution of gardens and ornamental horticulture, occupies critical role.The orchid kind successfully realizing Excised Embryos comprises HERBA DENDROBII, dendrobium candidum, great Hua all ages orchid, the color stem of noble dendrobium, Platanthera, short all ages orchid etc.The roxburgh anoectochilus terminal bud protocorms Excised Embryos system survival rate of current foundation is lower, is also not enough to the demand meeting production and scientific research.
Summary of the invention
The shortcoming of prior art in view of the above, the object of the present invention is to provide a kind of method of optimization roxburgh anoectochilus terminal bud protocorms vitrification ultra-low temperature preservation effect newly, be difficult to realize to overcome the medium-term and long-term preservation of plant germplasm resource in prior art, and the plant of Excised Embryos or the low shortcoming of its organized renewing growth rate.
To achieve these goals or other objects, the present invention is achieved by the following technical solutions.
Optimize a method for roxburgh anoectochilus terminal bud protocorms vitrification ultra-low temperature preservation effect, the method adopting vitrification ultra-low temperature to preserve is preserved roxburgh anoectochilus terminal bud protocorms, and concrete steps are as follows:
1) preculture: roxburgh anoectochilus terminal bud protocorms is placed on pre-culture medium, preculture 1 ~ 5 day in illumination box;
2) load liquid process: under room temperature, roxburgh anoectochilus terminal bud protocorms immersion treatment in loading liquid was removed loading liquid after 40 ~ 60 minutes;
3) vitrification solution process: use vitrification solution to soak dehydration processing roxburgh anoectochilus terminal bud protocorms 40 ~ 60 minutes at 0 ~ 25 DEG C;
4) Liquid nitrogen storage: maintenance roxburgh anoectochilus terminal bud protocorms is immersed in the state in vitrification solution, and Excised Embryos in the liquid nitrogen be placed on;
Described vitrification solution is the glass freezing protection liquid containing 0.1 ~ 0.5g/L graphene quantum dot.
Described MS culture fluid contains 1900mg/LKNO 3, 1650mg/LNH 4nO 3, 170mg/LKH 2pO 4, 370mg/LMgSO 47H 2o, 440mg/LCaCl 22H 2o, 37.3mg/LNa 2-EDTA, 27.8mg/LFeSO 47H 2o, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L puridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH 3bO 3, 22.3mg/LMnSO 44H 2o, 8.6mg/LZnSO 47H 2o, 0.25mg/LNa 2moO 42H 2o, 0.025mg/LCuSO 45H 2o, 0.025mg/LCoCl 26H 2o, surplus is water.The pH of described MS culture fluid is 5.8.
Preferably, above-mentioned steps 1) described in pre-culture medium be the MS solid culture medium of the sorbierite containing 60g/L; Described MS solid culture medium contains 1900mg/LKNO 3, 1650mg/LNH 4nO 3, 170mg/LKH 2pO 4, 370mg/LMgSO 47H 2o, 440mg/LCaCl 22H 2o, 37.3mg/LNa 2-EDTA, 27.8mg/LFeSO 47H 2o, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L puridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH 3bO 3, 22.3mg/LMnSO 44H 2o, 8.6mg/LZnSO 47H 2o, 0.25mg/LNa 2moO 42H 2o, 0.025mg/LCuSO 45H 2o, 0.025mg/LCoCl 26H 2o, 30g/L sucrose, 10g/L agar powder, surplus is water.The pH of described MS solid culture medium is 5.8.
Preferably, above-mentioned steps 1) described in the method for cultivating on MS solid culture medium be: by be placed under roxburgh anoectochilus terminal bud protocorms room temperature on pre-culture medium cultivate 1 ~ 5 day.
More preferably, cultivate being placed under roxburgh anoectochilus terminal bud protocorms room temperature on pre-culture medium 5 days.
Preferably, described loading liquid is for containing 1 ~ 2mol/L glycerine, 0.3 ~ 0.5mol/L sucrose and 5 ~ 10mmol/LKNO 3mS culture fluid.
Preferably, described loading liquid is for containing 2mol/L glycerine, 0.4mol/L sucrose and 10mmol/LKNO 3mS culture fluid.
Preferably, above-mentioned steps 2) in, under room temperature, roxburgh anoectochilus terminal bud protocorms immersion treatment in loading liquid was removed loading liquid after 40 minutes;
Preferably, above-mentioned steps 3) in, at 0 DEG C, use vitrification solution to soak dehydration processing roxburgh anoectochilus terminal bud protocorms 50 minutes.
Preferably, described vitrification solution is for containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1 ~ 0.5g/L graphene quantum dot.
Preferably, described vitrification solution is for containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1 ~ 0.3g/L graphene quantum dot.
Thawing and cultural method again of a kind of roxburgh anoectochilus terminal bud protocorms, described roxburgh anoectochilus terminal bud protocorms is the roxburgh anoectochilus terminal bud protocorms adopting method as described above to preserve, described roxburgh anoectochilus terminal bud protocorms thaw and again cultural method for roxburgh anoectochilus terminal bud protocorms is taken out from liquid nitrogen, first water-bath is thawed, then wash with cleaning solution after removing vitrification solution, finally proceed to renewal cultivation in recovery media.
Preferably, the condition that water-bath is thawed is the 60 ~ 120s that thaws in the water-bath of 30 ~ 40 DEG C.
More preferably, the condition that water-bath is thawed is the 60s that thaws in the water-bath of 40 DEG C.
Preferably, described cleaning solution is for containing 1.0 ~ 1.5mol/L sucrose and 5 ~ 10mmol/LKNO 3mS culture fluid.
Preferably, described cleaning solution is for containing 1.2mol/L sucrose and 10mmol/LKNO 3mS culture fluid.
Washing process is: at room temperature soaked 10 ~ 30 minutes by roxburgh anoectochilus terminal bud protocorms with cleaning solution.
More preferably, washing process is: with cleaning solution at room temperature by roxburgh anoectochilus terminal bud protocorms process 20 minutes, changes cleaning solution every 10 minutes.
Preferably, described recovery media is the MS solid culture medium containing 1 ~ 2mg/L6-benzyladenine, 0.1 ~ 0.5mg/L methyl α-naphthyl acetate and 30g/L sucrose.
Preferably, described recovery media is the MS solid culture medium containing 2mg/L6-benzyladenine, 0.1mg/L methyl α-naphthyl acetate and 30g/L sucrose.
Roxburgh anoectochilus terminal bud protocorms described in the present invention can be that method conventional in prior art obtains.Such as, preferably, preparation method's bibliography of roxburgh anoectochilus terminal bud protocorms: hybrid cymbidium ' phantom ' tissue culture regeneration Establishing and Vitro Preservation, Liu Peipei, Beijing Forestry University's master thesis, 2008.
More preferably, described vitrification solution is for containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.3g/L graphene quantum dot.
More preferably, in order to prove the preservation effect of method in the present invention, relative survival is adopted to add up, particularly, utilize TTC (being abbreviated as TTC) legally constituted authority meter roxburgh anoectochilus terminal bud protocorms relative survival after 30 days at renewal cultivation, and be aided with the observation of fluorescein(e) diacetate (being abbreviated as FDA) decoration method.
According to nano science principle, in cryoprotector, add viscosity and thermal conductivity that nano material effectively can improve cryoprotector, change the formation situation of ice crystal, reduce injury to cell.The present invention preserves under vitrification ultra-low temperature condition roxburgh anoectochilus terminal bud protocorms, first carry out preculture, then successively with loading liquid, vitrification solution process, finally Excised Embryos in liquid nitrogen, wherein vitrification solution is again for the addition of the vitrification solution of graphene quantum dot, other techniques in the interpolation fitting method of this allogenic material, effectively can improve the preservation effect of roxburgh anoectochilus terminal bud protocorms.According to store method disclosed by the invention, concentration is adopted to be that the graphene quantum dot of 0.1 ~ 0.5g/L is when using as allogenic material, recovery percentage after the preservation of roxburgh anoectochilus terminal bud protocorms vitrification ultra-low temperature is all significantly increased, method disclosed in the present invention is remarkable to the preservation effect optimization of roxburgh anoectochilus terminal bud protocorms, plays facilitation by adding graphene quantum dot as allogenic material to the preservation of plant vitrification ultra-low temperature.
Accompanying drawing explanation
Fig. 1 is the FDA stained photographs of roxburgh anoectochilus terminal bud protocorms Excised Embryos restoration ecosystem in experimental group and control group in embodiment.FDA can enter in living cells protoplast and produce fluorescence, can as judging cell mark anyway.
Have in Fig. 1 and be left-to-rightly respectively the hybrid cymbidium protocorms after the hybrid cymbidium protocorms after control group Excised Embryos, the hybrid cymbidium protocorms after experimental group 1 Excised Embryos, the hybrid cymbidium protocorms after experimental group 2 Excised Embryos, experimental group 3 Excised Embryos.As shown in Figure 1, brightness larger expression fluorescence intensity is stronger, and cell viability is higher.
Embodiment
Below by way of specific instantiation, embodiments of the present invention are described, those skilled in the art the content disclosed by this specification can understand other advantages of the present invention and effect easily.The present invention can also be implemented or be applied by embodiments different in addition, and the every details in this specification also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.
Roxburgh anoectochilus terminal bud protocorms used in embodiment by Stem tip induction, concrete grammar with reference to " hybrid cymbidium ' phantom ' tissue culture regeneration Establishing and Vitro Preservation ", Liu Peipei, Beijing Forestry University's master thesis, 2008.
The formula of the experiment reagent used in embodiment is as follows:
1) MS culture fluid is: MS culture fluid contains 1900mg/LKNO 3, 1650mg/LNH 4nO 3, 170mg/LKH 2pO 4, 370mg/LMgSO 47H 2o, 440mg/LCaCl 22H 2o, 37.3mg/LNa 2-EDTA, 27.8mg/LFeSO 47H 2o, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L puridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH 3bO 3, 22.3mg/LMnSO 44H 2o, 8.6mg/LZnSO 47H 2o, 0.25mg/LNa 2moO 42H 2o, 0.025mg/LCuSO 45H 2o, 0.025mg/LCoCl 26H 2o, surplus is water, and the pH of described MS culture fluid is 5.8.
2) MS solid culture medium is: MS solid culture medium contains 1900mg/LKNO 3, 1650mg/LNH 4nO 3, 170mg/LKH 2pO 4, 370mg/LMgSO 47H 2o, 440mg/LCaCl 22H 2o, 37.3mg/LNa 2-EDTA, 27.8mg/LFeSO 47H 2o, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L puridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH 3bO 3, 22.3mg/LMnSO 44H 2o, 8.6mg/LZnSO 47H 2o, 0.25mg/LNa 2moO 42H 2o, 0.025mg/LCuSO 45H 2o, 0.025mg/LCoCl 26H 2o, 30g/L sucrose, 10g/L agar powder, surplus is water, and the pH of described MS solid culture medium is 5.8.
3) pre-culture medium is the MS medium containing 60g/L sorbierite.
4) loading liquid is: containing 2mol/L glycerine, 0.4mol/L sucrose and 10mmol/LKNO 3mS culture fluid.
5) vitrification solution is: containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1 ~ 0.5g/L graphene quantum dot.
6) cleaning solution is for containing 1.2mol/L sucrose and 10mmol/LKNO 3mS culture fluid.
7) recovery media is the MS solid culture medium containing 2mg/L6-benzyladenine, 0.1mg/L methyl α-naphthyl acetate and 30g/L sucrose.
Embodiment
1) by roxburgh anoectochilus terminal bud protocorms illumination preculture 5 days on the MS solid culture medium containing 60g/L sorbierite;
2) go to and to load in liquid soaking at room temperature process 40 minutes;
3) dehydration processing is proceeded in vitrification solution under 0 DEG C of condition 50 minutes;
4) liquid nitrogen Excised Embryos is finally placed in.
Step 3) terminate after, without the need to removing vitrification solution, directly the roxburgh anoectochilus terminal bud protocorms be soaked in vitrification solution is placed in liquid nitrogen Excised Embryos.
According to above-mentioned steps, roxburgh anoectochilus terminal bud protocorms is divided into experimental group and control group.
Wherein, the graphene quantum dot respectively containing 0.1g/L, 0.3g/L, 0.5g/L in the vitrification solution of experimental group.
Particularly, the graphene quantum dot containing 0.1g/L in the vitrification solution of experimental group group 1.
Graphene quantum dot containing 0.3g/L in the vitrification solution of experimental group group 2.
Graphene quantum dot containing 0.5g/L in the vitrification solution of experimental group group 3.
In control group, difference is not add graphene quantum dot in vitrification solution, and other are identical with experimental group.
Preserve in liquid nitrogen after 1 hour and take out, put into 40 DEG C of water-baths fast, thaw 60s, and frequently shake gently; Absorbed by vitrification solution, add cleaning solution, room temperature treatment 20 minutes, changed once washing liquid every 10 minutes; Roxburgh anoectochilus terminal bud protocorms after washing moves on to after recovery media cultivates 30 days, the relative survival of roxburgh anoectochilus terminal bud protocorms in comparative experiments group and control group.
The relative survival of the roxburgh anoectochilus terminal bud protocorms of experimental group and control group is in table 1.
Table 1
Experimental result
As shown in Table 1, be that the graphene quantum dot of 0.1g/L, 0.3g/L and 0.5g/L adds in the vitrification solution of roxburgh anoectochilus terminal bud protocorms Excised Embryos by concentration, make roxburgh anoectochilus terminal bud protocorms recovery percentage bring up to 8.67%, 10.72% and 6.47% respectively by 4.52%, concrete effect is shown in Fig. 1.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.

Claims (2)

1. optimize a method for roxburgh anoectochilus terminal bud protocorms vitrification ultra-low temperature preservation effect, it is characterized in that, the method adopting vitrification ultra-low temperature to preserve is preserved roxburgh anoectochilus terminal bud protocorms, and concrete steps are as follows:
1) preculture: roxburgh anoectochilus terminal bud protocorms is placed on pre-culture medium, preculture 5 days in illumination box;
2) load liquid process: under room temperature, roxburgh anoectochilus terminal bud protocorms immersion treatment in loading liquid was removed loading liquid after 40 minutes;
3) vitrification solution process: use vitrification solution to soak dehydration processing roxburgh anoectochilus terminal bud protocorms 50 minutes at 0 ~ 25 DEG C;
4) Liquid nitrogen storage: maintenance roxburgh anoectochilus terminal bud protocorms is immersed in the state in vitrification solution, and is placed on Excised Embryos in liquid nitrogen;
Described vitrification solution is the glass freezing protection liquid containing 0.1 ~ 0.5g/L graphene quantum dot; Described roxburgh anoectochilus terminal bud protocorms pre-culture medium is the MS solid culture medium containing 40 ~ 80g/L sorbierite;
Described loading liquid is for containing 1 ~ 2mol/L glycerine, 0.3 ~ 0.5mol/L sucrose and 5 ~ 10mmol/LKNO 3mS culture fluid;
Described vitrification solution is for containing 300g/L glycerine, 150g/L ethylene glycol, 150g/L dimethyl sulfoxide (DMSO), the MS culture fluid of 0.4mol/L sucrose and 0.1 ~ 0.5g/L graphene quantum dot.
2. the thawing and cultural method again of a roxburgh anoectochilus terminal bud protocorms, described roxburgh anoectochilus terminal bud protocorms is the roxburgh anoectochilus terminal bud protocorms adopting method as claimed in claim 1 to preserve, described roxburgh anoectochilus terminal bud protocorms thaw and again cultural method for roxburgh anoectochilus terminal bud protocorms is taken out from liquid nitrogen, first water-bath is thawed, then wash with cleaning solution after removing vitrification solution, finally proceed to renewal cultivation in recovery media;
The condition that water-bath is thawed is the 60 ~ 120s that thaws in the water-bath of 30 ~ 40 DEG C;
Described cleaning solution is for containing 1.0 ~ 1.5mol/L sucrose and 5 ~ 10mmol/LKNO 3mS culture fluid washing, washing process is: with cleaning solution at room temperature by roxburgh anoectochilus terminal bud protocorms soak 10 ~ 30 minutes;
Described recovery media is the MS solid culture medium containing 1 ~ 2mg/L6-benzyladenine, 0.1 ~ 0.5mg/L methyl α-naphthyl acetate and 30g/L sucrose.
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CN105557514B (en) * 2015-12-14 2018-11-13 李操 A kind of Cryopreservation of bud germ plasm resource
CN108812308A (en) * 2018-05-21 2018-11-16 上饶师范学院 A method of improving early pears stem apex encapsulation- vitrification method cryopreservation effect

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